Over 100 different types of epithelial cells perform essential vectorial functions required for homeostasis and survival of the organism. A key property underlying these functions is polarity, i.e. the ability of epithelial cells to localize different transporters, channels and hormone receptors to opposite (apical and basolateral) domains of their plasma membrane (PM). The experiments proposed will utilize state of the art molecular, biochemical and microscopic techniques to elucidate fundamental mechanisms involved in epithelial polarization. The proposed experiments follow up on our group's recent finding that clathrin plays a broad role of clathrin in basolateral protein sorting and that some epithelia lack the basolateral sorting adaptor AP-1B. Specific aim 1 will investigate in detail the roles of clathrin adaptors AP-1A and AP-1B, two major basolateral sorting adaptors, in carrier assembly by quantitative 4D 3-color live imaging analysis. We will search for trafficking machinery engaged by AP-1A and AP-1B using a proteomics approach developed by Bernard Hoflack (Dresden). Specific aim 2 will investigate the role of clathrin adaptors AP-2, AP-3, AP-4 and GGAs in polarized trafficking, using siRNA and ad hoc trafficking assays. The experiments will focus both on MDCK cells and on kidney proximal tubule epithelium, which lacks AP-1B. Specific aim 3 will investigate the role of Golgins and rabs in polarized trafficking, combining Golgin knock-down and inhibition of candidate rabs with trafficking assays for a panel of apical and basolateral cargos. We will test the hypothesis that TGN Golgins, elongated coil-coil molecules that bind membranes via rabs, mediate highly dynamic physical interactions between TGN and recycling endosomes that are required for efficient polarized sorting. Specific aim 4 will characterize the specific steps regulated by rabs 6,8,10, 11,17,25 in polarized trafficking, using ad hoc assays to explore these steps. The studies proposed will contribute to a deeper understanding of epithelial trafficking mechanisms, which, in turn, is likely to help understand and develop cures for human diseases.